This invention relates to an automatic recording fluorometer/densitometer.
Zonal electrophoresis has been successfully used in clinical laboratories. Electrophoresis is a separative procedure based on the phenomenon that electrically-charged molecules or particles will migrate through a solution or gel in response to an externally applied voltage gradient. Many biological molecules, especially proteins, carry a net electric charge in solution which makes them susceptible to electrophoretic forces.
In the zonal electrophoresis technique, a small volume of biological fluid (e.g., blood serum, cerebrospinal fluid, etc.) is applied at one spot on a buffer-saturated membrane or thin-layer medium. When a voltage difference is established across the ends of the medium, different molecular species in the sample migrate with different velocities, and with time, resolve into a series of distinct bands or spots. The desired molecular bands can be visualized by direct staining with specific dyes, or in the case of certain enzymatic samples, by applying chemical substrates that become catalytically converted to colored or fluorescent products. Deviations from normal in the intensity or mobility of the bands revealed by a given test are associated with biochemical abnormalities that may indicate a particular pathology.
Electrophoresis is often effective in separating nearly identical biomolecules, since minor differences in charge and molecular conformation may result in noticeably different electrophoretic mobilities. Similar electrophoretic procedures can be applied to a wide variety of clinical tests, since the specificity of a given test is largely determined by the dyes or reagents used in the final processing. The simplicity, rapidity and versatility of zonal electrophoresis, as well as its general effectiveness, make it an attractive basis for clinical determinations that require the separation of biomolecules.
U.S. Pat. Nos. 3,479,265 and 3,635,808 disclose thin film agarose plates which can be used as the electrophoretic medium. The thin film plates of these patents are particularly convenient for handling and storage.
Electrophoresis preparations or samples can be grouped into two categories, densitometric and fluorometric. Densitometric (sometimes called colorimetric) samples have bands that absorb visible light and are thus observable in normal room light. Fluorometric samples absorb ultraviolet light and fluoresce, emitting light at visible wavelengths. Thus fluorometric samples cannot be seen in normal room light but must be excited with ultraviolet light to be observed.
Qualitative clinical evaluation of electrophoresis samples can be done by visual inspection, i.e., gross abnormalities can be detected in this way. However, the present state-of-the-art in clinical medicine typically requires a more critical quantitative evaluation.
Quantitative evaluation of electrophoresis preparations requires the generation of an optical density profile or fluorescence intensity profile of the sample, whichever is appropriate.
The optical density profile is partitioned into individual peaks each representing a band on the electrophoretic separation. Adjacent peaks are separated by recognizing and selecting a valley. Integration of the area under each individual peak and computation of the peak area as a percentage of the total area under the profile represents, for example, the distribution of certain proteins in blood serums. Norms for such distributions have been established, and deviation from these norms is of diagnostic significance. The percentage numbers are sometimes multiplied by a "scale factor" so that the results are in units of protein concentration or enzyme activity, rather than percent of total.
One instrument for automatically making analyses of the aforementioned type is described in U.S. Pat. No. 3,706,877.
In addition to automatically recording the concentration distributions of the film, it is desirable to directly view the films. Fluorometric iso-enzyme samples have bands of iso-enzymes dispersed along the length of any given sample which cannot be seen in room light. The sample must be shielded from visible light and illuminated with ultraviolet light in order to see the iso-enzyme bands.
Currently available viewers for fluorescing samples usually make use of a hand held source of ultraviolet radiation in a darkened room or a sample is inserted into a box and viewed through a port with the sample being viewed from the same side that it is illuminated. Inherent in this arrangement is both reduced intensity and reduced uniformity since bulbs must be placed outside the field of view and relatively distant from the sample. State of the art fluorescent viewing devices do not provide operator protection from exposure to ultraviolet radiation.